Display methodologies have proven invaluable for the discovery, production, and optimization of proteins and peptides in a variety of biotechnological applications. Various approaches including phage display (Smith, G. P. (1985) Science, 228, 1315-1317), mRNA (Wilson et al. (2001) Proc. Natl. Acad. Sci. USA, 98, 3750-3755) and DNA display (Yonezawa et al. (2003) Nucleic Acids Res., 31, e118), ribosome display (Hanes, J. & Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA, 94, 4937-42), eukaryotic virus display (Bupp, K. & Roth, M. J. (2002) Mol. Ther., 5, 329-335; Muller et al. (2003) Nat. Biotechnol., 21:1040-1046), yeast display (Boder, E. T. & Wittrup, K. D. (1997) Nat. Biotechnol., 15, 553-557), and bacterial display (Lu et al. (1995) Biotechnology (NY), 13, 366-372) have been developed to screen diverse molecular repertoires for desired activities. In particular, bacterial display libraries have enabled antibody affinity maturation (Daugherty et al. (2000) Proc. Natl. Acad. Sci. USA, 97, 2029-2034), the discovery of protein binding peptides (Bessette et al. (2004) Protein Eng. Des. Sel., 17, 731-739), cell-specific ligands (Dane et al. (2006) J. Immunol. Methods, 309, 120-129; Nakajima et al. (2000) Gene, 260, 121-131), and the identification of optimal protease substrates (Boulware, K. T. & Daugherty, P. S. (2006) Proc. Natl. Acad. Sci. USA, 103, 7583-7588). One of the key advantages of bacterial surface display is the ability to use flow cytometry for quantitative screening of the libraries, allowing for real-time analysis of binding affinity and specificity to optimize the screening process (Wittrup, K. D. (2001) Curr. Opin. Biotechnol., 12, 395-399). Additionally, the ease of genetic manipulation, high transformation efficiency, and rapid growth rate make E. coli a well-suited host for display. A broad range of bacterial surface display systems have been developed allowing for insertional or terminally fused peptides and proteins to be displayed on the cell surface. Several outer membrane proteins and cellular appendage proteins have been used to present polypeptides as insertional fusions (Bessette et al. (2004) Protein Eng. Des. Sel., 17, 731-739; Charbit et al. (1986) Embo J., 5, 3029-3037; Taschner et al. (2002) Biochem. J., 367, 393-402). The ice nucleation protein (Jung et al. (1998) Nat. Biotechnol., 16, 576-580), intimins (Christmann et al. (1999) Protein Eng., 12, 797-806), and LppOmpA (Francisco et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 2713-2717) have been used to display proteins on the C-terminus of a transmembrane scaffold while N-terminal display has been accomplished using autotransporters IgA1 protease and EstA (Maurer et al. (1997) J Bacteriol, 179, 794-804).
Recently, a unique bacterial display scaffold was developed that allows for N- and/or C-terminal display from a circularly permuted variant of outer membrane protein OmpX (CPX) (Rice et al. (2006) Protein Sci., 15, 825-836). This scaffold enables display of peptides on both termini, but with reduced efficiency when compared to that obtained using insertions into OmpX. Reduced membrane localization of CPX may result from slower folding rates and reduced stability that has been described previously for circularly permuted proteins (Heinemann, U. & Hahn, M. (1995) Prog. Biophys. Mol. Biol., 64, 121-143). Regardless, reduced display efficiency requires longer induction times to achieve sufficient display for screening by FACS. Importantly, inefficient display can create an undesired selection pressure resulting in growth biases, reduced viability, or differing levels of passenger localization on the cell surface. As a result, screening based upon cell fluorescence can favor passengers most efficiently localized to the surface, rather than passengers enhanced for the properties of interest (e.g., binding affinity).
Thus, there remains a need for additional vectors for bacterial cell display and methods that would more effectively display proteins and peptides.